How the human pathogen Mycoplasma pneumoniae attenuates virulence factor production during growth as biofilm towers, and how this attenuation favors chronic respiratory disease, including both pneumonia and asthma, are unknown. Our long-term goal is to understand the processes that are essential for establishing and maintaining M. pneumoniae infection. The overall objective of this application is to understand how M. pneu- moniae biofilm towers chronically infect respiratory epithelial host cells at an air-liquid interface (ALI). Our central hypothesis is that the reduction in virulence factor production by M. pneumoniae bacteria in biofilm towers is signaled by decreased O2 levels and results in minimization of both defensive responses and damage to the epithelium. The rationale underlying these experiments is that once the extent to which damage is limited by changes to M. pneumoniae during biofilm tower growth and maintenance is understood, approaches that counter the scheme of limiting host responses could be developed as therapeutic agents to reduce morbidity associated with chronic M. pneumoniae infection. We plan to test our central hypothesis by pursuing the follow- ing two specific aims: 1) characterize the development and impact of M. pneumoniae biofilms in a respiratory epithelial air-liquid interface tissue culture model; and 2) determine the role of O2 in regulating M. pneumoniae gene expression and steady-state levels of virulence-associated proteins in vitro. For the first aim, we will char- acterize the impact of M. pneumoniae biofilm towers on airway epithelia in an ALI tissue culture model using microscopy, transcriptomics of the host cells, and measurements of epithelial barrier function like transepithelial electrical resistance, ciliation, and localization of the ZO-1 tight junction protein, comparing these effects to those produced by CARDS toxin and H2O2, toxic molecules whose production is attenuated in M. pneumoniae during biofilm tower growth. For the second aim, prompted by preliminary data about the decreased activity of the H2O2-producing enzyme glycerol 3-phosphate oxidase in M. pneumoniae grown in low O2, we will investigate the bacterial response to growth in 10% O2 at the levels of transcription a global level, and, for select virulence- associated proteins, steady-state abundance. The proposed research is innovative, in our opinion, because it represents a substantive departure from the status quo by focusing on how M. pneumoniae both responds to and impacts its environment within a biofilm context, using biofilm tower growth in an ALI airway epithelial model and studying gene regulation in a biofilm environment. This contribution will be significant because it is expected to direct future research efforts toward development of therapeutic intervention strategies targeting M. pneu- moniae biofilms. Additionally, the work in this AREA proposal will provide training in fundamental microbio- logical, transcriptomic,...